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Abstract Present study aimed at the isolation and characterization of bacterial pathogens from stool samples of diarrhoeal patients and evaluation of their antibacterial susceptibility pattern against commonly used antibiotics. Samples were collected from Divisional Headquarter (DHQ) and Allied Hospital, Faisalabad. Differential and selective media were used to isolate bacterial pathogens which were identified through cultural characteristics, microscopy and biochemical tests. Disc diffusion assay was done using Muller Hinton agar medium and Minimum Inhibitory Concentration (MIC) was determined using broth dilution method against isolated pathogens. According to the results, all 141 (100%) samples were positive for some bacteria. Frequently of occurrence was; Bacillus cereus (65.95%), E. coli (48.51%), Salmonella typhi (27.65%), Pseudomonas aeruginosa (8.51%) and Staphylococcus aureus (4.25%). Antibacterial activity of used antibiotics i.e. Ofloxacin, Gentamicin, Amoxicillin, Cefotaxime against B. cereus was 33.447±3.429, 22.500±3.110, 23.277±3.059 and 12.617±2.889, against E. coli 11.701±2.082, 13.448±2.025, 3.382±1.985 and 0.886±0.179, against Salmonella typhi 25.00±6.130, 14.692±4.786, 7.30±3.72 and 0.1601±0.0256, against Pseudomonas aeruginosa 15.583±3.260, 15.167±2.552, 0.00±0.00, 0.00±0.00 and against Staphylococcus aureus 28.00±3.22, 12.333±2.160, 5.83±4.71 and 0.00±0.00, respectively. Pseudomonas aeruginosa showed resistance against Amoxicillin and Cefotaxime whereas Staphylococcus aureus was found resistant against Cefotaxime. Statistical analysis through one way ANOVA revealed that Ofloxacin and Gentamicin had significant (p<0.05) activity against all isolates as compared to the other antibiotics used in this study. Keywords: Diarrhoea, Bacterial pathogens, Antibacterial susceptibility, Disc diffusion assay, Minimum Inhibitory Concentration 1 Introduction One billion people have poor excess to water and about 2.6 billion lack basic sanitation facilities worldwide [1]. Ten to eleven million children die before the age of five in low and middle income countries [2]. Among many other infectious diseases, diarrohea is an important cause of morbidity and mortality in human. There has been much less progress in reducing diarrohea over past decade and 21% mortality rate was estimated in the children of less than five years of age throughout the world [3]. Approximately 2 million children die in low and middle income countries only due to diarrohea [4]. It accounts for 12% of all deaths due to infectious disease in the world. Approximately 90% of diarrhoeal cases in children are in low and middle income countries [5]. Among different types of diarrhoea, acute watery diarrhoea is more common and has morbidity rate of 80% and mortality rate of 50% in children [6]. Persistent diarrhoea has the morbidity rate of only 10% but have increased risk of death due to malnutrition. Globally, persistent diarrhoea has mortality rate of 35%, but Asia is proved to be more responsible for more than half the diarrhoeal deaths [7]. Diarrhoea can be caused by a variety of microorganisms that may be viral, bacterial, and protozoan. Most diarrhoea causing microbes have medium to low virulence. Shigella in few numbers can cause symptomatic disease [8]. High concentration of Vibrio cholerae requires for causing disease in healthy individual. The other bacterial etiological agents are Escherichia coli, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella and Shigella serotypes. The dangerous form of the diarrhoea is cholera, caused by Vibrio cholerae. The fluid loss in this condition if goes untreated leads to death [9]. Dysentery is the passing of stools with blood. Common cause of the dysentery is Shigella spp. and amebiosis [10, 11]. To find diarrhoeal causative agent from diarrhoea episodes is difficult even after under controlled 2 conditions as pathogens are not found in half of the cases in modern hospitals [12]. The viruses associated with acute gastroenteritis are Rotavirus, Norovirus, Enteric Adenovirus, Calicivirus and Astrovirus and among these the Rotavirus is the most common cause of severe diarrhoea [13]. Route of the parasite transmission is either through food or water. Parasites like Cyclospora cayetanenisis, Cryptosporidium, Entamoeba histolytica and Giardia lamblia also cause diarrhoea [14]. There are two pathophysiological mechanisms by which bacteria cause disease. Either they produce toxins that affect the intestinal fluid or by the intrusion of the microbes. Cholera toxin causes the fluid secretion. Transmission route of the bacteria is by the fecal-oral route by means of unhygienic water, food, person to person interaction or by the direct contact. Diarrhoea is transmitted by means of domestic storage containers. Protection against diarrhoeal agents also relies on the microflora of the gastro-intestinal tract. Most of the pathogens are killed by the hydrochloric acid. People having low level of gastric acid are more prone to contact diarrhoea than others. Interrelated factors like family formation, sanitizing conditions, availability of clean water and food, household environment, society trends, policies and individual behavior also affect the rate of morbidity and mortality by the diarrhoea [15]. Antibiotics are not generally recommended to children with infectious causes [6]. But antibiotics are given in case of having cholera and dysentery [16]. Zinc is used to reduce the severity and duration of the disease and lower the incidence of diarrhoea in the following 2-3 months [17]. Now, it is recommended to use zinc as soon as possible for the treatment of diarrhoea [18]. Most severe form is the persistent or chronic diarrhoea, for its treatment initially correction of malnutrition is required [19]. Rehydration is essential by maintaining the electrolytes. Symptomatic relief is the second priority [20]. In 1979, Oral rehydration therapy 3 (ORT) was introduced and became an important task for the CDD program (Control of Diarrhoeal Disease). ORT contains sodium, a carbohydrate and water [21]. Keeping in view the importance of the disease and associated losses, the present study was conducted to know the occurrence of the bacterial pathogens responsible for diarrhoea in District Faisalabad Punjab Pakistan and to engender information regarding the effectiveness of commonly used antibiotic against diarrhoea. It is hoped that the findings will be helpful to establish an effective treatment and thus better control of diarrhoea in future. Materials and Methods Sample collection A total of 141 stool samples of either sex and from different age groups were collected from District Headquarter Hospital (DHQ) and Allied Hospital Faisalabad Punjab Pakistan. Samples were obtained in sterile containers, properly labelled and transported in the Carry-Blair Medium (Oxoid, UK) along with complete data sheet and under aseptic conditions to Postgraduate Research Laboratory of Department of Microbiology, Govt College University Faisalabad Pakistan [22]. Isolation and purification of bacterial pathogens The stool samples were inoculated first on Nutrient agar (Scharlau, Spain) plates by swabbing and incubated at 37°C for 24 h. For purification, the different colonies from initial growth were streaked on MacConkey’s agar (Oxoid, UK) and Salmonella Shigella agar (Oxoid, UK) plates and incubated at 37°C for 24 h [23]. Identification and biochemical characterization Identification was done on the basis of cultural characteristics on different media and microscopic examination of stained smears through Gram staining procedure. Biochemical 4 characterization was done using catalase, methyl red, indole production, Voges Proskauer, coagulase, oxidase and triple iron suger tests [24, 25, 26]. Antibiotic susceptibility testing a. Disc diffusion method The agar disc diffusion method (Kirby-Bauer method) was done using Muller-Hinton agar medium (Oxoid, UK). The antibacterial susceptibility of bacterial isolates was determined against selected commonly used antibiotics including Ofloxacin, Gentamicin, Amoxicillin and Cefotaxime. Turbidity of inoculum was adjusted to 0.5 McFarland and diameter of Zone of inhibition were measured in mm [27, 28]. b. Minimum Inhibitory Concentration (MIC) The MIC is the lowest concentration of the antibiotics which prevents visible growth of microbes. It was measured by Micro-broth dilution method using 96-well microtitration plate. Muller-Hinton broth (Oxoid, UK) was used with phenol red (0.01%) as an indicator [29]. Statistical analysis The data was statistically analyzed by computing coefficient of variance and comparing the mean with standard deviation and One-way Analysis of Variance (ANOVA) using the Minitab 15. Results Sample distribution Out of total 141 diarrhoeal stool samples collected, 100% pathogen response rate was found and different bacteriological agents responsible for diarrhoea from the same specimen were isolated. The patients were categorized into three age groups from infants to the older ones. First group (G1), was from 1 day to 5 years (n=60) second group (G2) aged between 5-30 years 5 (n=45) and third group (G 3) from 30 years to onwards (n=36). The 57 (40.42%) samples were from male patients and 84 (59.58%) were of females. Occurrence of bacterial isolates Single pathogen was detected in only 20 (14.18%) samples whereas combinations were found in 121 (85.81%) samples. Bacillus cereus and E. coli were the most frequently detected pathogens followed by the Salmonella typhi, Pseudomonas aeruginosa, and Staphylococcus aureus. The percentage occurrence of isolated pathogens was; Bacillus cereus (43%), E. coli (31%), Salmonella typhi (18%), Pseudomonas aeruginosa (5%) and Staphylococcus aureus (3%) (Figure 1). Identification of bacterial isolates a. Colony characteristics The growth of E. coli was observed as pink, shiny, smooth, round colonies with raised surface and varying growth pattern on MacConkey’s agar. Bacillus Cereus has large, smooth, pink colonies with mousy smell on MacConkey’s agar. Lactose non fermenter colonies on the MacConkey’s agar and central black, small size colonies with smooth to rough in appearance on the Salmonella-Shigella agar were identified as Salmonella spp. Pseudomonas aeruginosa was found to be Non lactose fermenter with colourless, irregular and round colonies having sweet odour on the MacConkey’s agar. Smooth, creamy and yellow colonies on the Staphylococcus 110 agar were observed for Staphylococcus aureus (Figure 2 a, b, c, d). b. Microscopic examination Gram’s staining was done to identify the bacterial isolates on the basis of their morphology, arrangement, and staining characters. Different isolates showed difference in these parameters which helped in their identification (Table 1 and Figure 3 a, b). 6 c. Biochemical characterization Different biochemical tests were used to confirm the bacterial isolates including methyl red, Voges Proskauer, catalase, indole production, coagulase and oxidase tests. The results are shown in Table 2 and Figure 4 a, b. Disc diffusion method The diameter of Zones of inhibition were measured in mm. Bacillus cereus showed the highest sensitivity against Ofloxacin with mean diameter of 33.447±SD and least sensitivity against Cefotaxime as 12.617±SD. E. coli revealed highest sensitivity against Gentamicin with 13.448±SD mean diameter and it was found resistant against Cefotaxime. Salmonella typhi exhibited the highest sensitivity against Gentamicin with diameter 13.448±SD whereas found resistant against Cefotaxime. Pseudomonas aeruginosa displayed highest sensitivity against Gentamicin with 15.583±SD mean diameter and indicated resistance against Cefotaxime and Amoxicillin. Staphylococcus aureus presented highest sensitivity against Ofloxacin with the 28.00±SD mean diameter and found resistant against Cefotaxime (Table 3 and Figure 5 a, b, c, d). Minimum Inhibitory Concentration (MIC) The MIC of different antibiotics was measured as the minimum concentration of the antibiotic that inhibited the visible growth of microorganisms. Statistically, B. cereus was highly sensitive against Metronidazole with the mean ± SD as (7.526±2.513) and E. coli against Ciprofloxacin with mean ± SD (2.491±0.770). Salmonella typhi was highly sensitive against Amoxicillin with the mean ± SD (2.362±0.792) wheras Pseudomonas aeruginosa showed highest sensitivity against Ofloxacin with the mean ± SD (7.916±2.574). Staphylococcus aureus was highly sensitive against Ciprofloxacin with the mean ± SD (2.340±0.854) (Table 4). 7 Discussion The present study was performed to determine the type and occurrence of bacterial pathogens involved in diarrhoeal cases from patients of both sex and different age groups in District Faisalabad Punjab Pakistan. The antibacterial susceptibility pattern of isolated bacteria was also studied against commonly used antibiotics. From the collected stool samples, bacterial pathogens like Bacillus cereus, E. coli, Salmonella, Staphylococcus aureus and Pseudomonas aeruginosa species were isolated and identified. Previous literature also reported the presence of similar type of bacteria in the stool specimens of diarrhoeal patients [30, 31, 32, 33]. Isolates were identified on the basis of colony characteristics on different selective media. Bacillus cereus showed pink colour colonies on the MacConkey’s agar due to the lactose fermentation. It forms large colonies because of adapting a wide range of environmental conditions [34, 35]. Salmonella typhi produced small black colour colonies on Salmonella Shigella agar as it produces the hydrogen sulphide [36]. Pseudomonas aeruginosa did not ferment lactose, so it appeared colourless on the MacConkey’s agar [37]. Staphylococcus aureus showed yellow colour colonies on the blood agar media [38]. Isolates were identified by the separation of the lactose fermenter and non-lactose fermenter bacteria using the novel approach presented by Khan et al. [39]. Isolates were confirmed on the basis of the results of biochemical tests. Bacillus cereus was confirmed in the ninety three (93) samples as Gram positive rods with MR-VP positive. Because of the accumulation of acids, pH of the medium drops due to which MR changes from yellow to red colour and a tinge of red colour in VP medium due to the production of non-acidic products [25, 26]. E. coli was identified in the sixty seven (67) samples with MR positive and VP negative as the addition of acids causing the pH of the medium falls due to which MR altered 8 from yellow to red colour and a shade of red colour in VP medium because of the production of non-acidic products. The indole production test was also positive as E. coli has the capability to generate indole from tryptophan by using an enzyme tryptophanase and was identified in presence of Kovac’s reagent [40]. Salmonella typhi was confirmed in the thirty nine (39) samples because of the indole negative and smooth, small and colourless colonies due to lack of H2S production on the Salmonella Shigella agar. Pseudomonas aeruginosa was identified in the twelve (12) stool samples due to the oxidase positive test indicating the presence of cytochrome enzyme. Staphylococcus aureus was confirmed in only six (06) samples showing catalase and coagulase test positive due to the presence of catalase enzyme that produce bubbles on reacting with the Hydrogen peroxide (H2O2) and releasing the products as water (H2O) and molecular oxygen (O2) [25]. The percentage occurrence of Bacillus cereus was the highest (65.95%) among all the isolates. Al-Khatib et al. [41] also reported similar results. Second most frequent isolated pathogen in all age groups was E. coli (48.51%). The reason for this may be the improper sanitization of utensils and unhygienic conditions in this region of the world. Third most frequently isolated pathogen was the Salmonella (27.65%) which was prevalent in G3 group and less common in G1 and G2. Frequency of occurrence of different bacterial pathogens in this work was almost similar to the work of Manikandan and Amsath [42] with approximately 10% variations. In this study, commercially available and commonly used four antibiotics (Ofloxacin, Gentamicin, Amoxicillin and Cefotaxime) were selected for antibacterial susceptibility testing. Ofloxacin and Gentamicin are broad-spectrum antibiotics that inhibit the protein synthesis [43, 44] wheras Amoxicillin and Cefotaxime inhibit peptidoglycan of the both Gram positive and 9 Gram negative cells [45, 46]. The overall results of disc diffusion assay revealed that all isolated pathogens showed statistically significant (p<0.05) sensitivity against Ofloxacin and Gentamicin wheras they were resistant to Amoxicillin and Cefotaxime. The B. cereus exhibited partial resistance against Amoxicillin and Cefotaxime, wheras it was sensitive to Gentamicin and Ofloxacin. The result coincides with the work of Turnbull [47] and Kiyomizu et al. [48]. E. coli showed resistance against Amoxicillin and Cefotaxime and sensitivity against Gentamicin and Ofloxacin. Salmonella presented sensitivity against Ofloxacin and resistance against Amoxicillin. A study conducted in the Bangladesh also revealed similar results [49]. Staphylococcus aureus showed sensitivity against Ofloxacin and resistance against Cefotaxime. Pseudomonas aeruginosa exhibited sensitivity against Ofloxacin and Gentamicin, whereas it was resistant against Amoxicillin and Cefotaxime. Conclusion Based on the overall result of present study, it was concluded that Bacillus cereus was the most frequently isolated organism from stool samples of diarrhoeal patients of both sex and all age groups followed by E. coli, Salmonella typhi, Pseudomonas aeruginosa and Staphylococcus aureus, respectively in District Faisalabad of Punjab Pakistan. The overall percentage of occurrence of diarrhoeal cases was more in the children and females than in males. 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